Screening

adapted from Johannes Rudolph

Goal

Use the FEI Tecnai T12 TEM to image negative stain.

Getting started

  1. Get trained by Garry Morgan (garry.morgan@colorado.edu)

  2. Reserve time using the online portal

  3. Log into the Windows server

  4. Check liquid nitrogen dewar and top off if necessary on right hand side of column

  5. Check if High Tension and Filament are on (buttons are yellow when on)

    • if not, turn them on (High Tension first, then Filament)

    • wait at least 5 min after turning on the filament to collect data

  6. Gun/Col vacuum should ideally read log 6

    • if sample has been inserted, often goes to log 12-14

    • if higher than that, seek Garry’s help

  7. Make sure the column valves are closed (button should be yellow)

  8. Make sure the beam is spread

    • open the viewing window and adjust using intensity dial

    • if column valves are closed, you won’t see anything

    • just set the C2 value on screen ~50%

Removing the sample holder

  1. Pull straight out, resisting vacuum, with other hand on plate

  2. Turn CW until it stops

  3. Re-grip and break seal of vacuum with thumb on plate

  4. Pull straight out the rest of the way, slowly

Loading grid into holder

Never touch bronze part of holder

  1. Use pin to ‘open door’

  2. Use tweezers to place grid into hole (can tap opposite end to position grid)

  3. Use pin to ‘close door’

  4. Give a gentle shake to make sure grid stays in there

Inserting sample holder

  1. Put pin at 2 o’clock (sample will be oriented vertically)

  2. Insert slowly until pin hits a stop

  3. Gently push holder while turning CW, another 1 -2 cm. Do this rapidly without a pause.

  4. Seal is created and red light/vacuum pump will go on (takes 1 min)

  5. Then rotate CCW to stop

  6. Vacuum will draw the holder in: guide it slowly so it doesn’t go too fast

Setting up acquisition software

  1. Open camera software (AMT icon) and move to other screen

  2. Start a new case study under File>New Case Study

    • make a new folder under your lab and user on the 192... server

    • make a new case study with the sample and grid number (make a new one for every grid)

Eucentric focusing (alpha wobbler adjustment)

  1. Open the column valves to let beam through the screen

  2. Adjust mag and beam intensity and center on a feature of the grid

  3. Press L1 on left control panel to start alpha wobbler

  4. Adjust Z-height on right control panel using light taps on plus/minus buttons until the feature no longer moves away from the center

  5. Press L1 again to turn off alpha wobbler

    • if it beeps while you are adjusting the alpha wobbler, then it has reached its limit.

    • first click “AB” to readjust the wobbler

    • then change Z-height to about 150 (without having the alpha wobbler turned on)

    • then try again to use the alpha wobbler by pressing L1

  6. Press Eucentric Focus on right panel

Adjusting the beam

  1. Insert objective using lever on side of scope

  2. Once in an area of interest, adjust mag and center beam with left track ball

  3. Also adjust intensity with dial on left control panel (should be 4-5 nanoAmps)

Using the camera

  1. Press Insert Camera on top right of acquisition panel (before inserting camera make sure the intensity is 4 - 5 nanoAmps to prevent burnout of camera)

  2. Click Live Image

  3. Adjust beam intensity, focus and mag as needed (generally need a mag of >40K to see particles)

  4. Click for final image and then right click on Save (this will close the final image and re-activate the live camera)

Pro-tip: focus/mag/ adjust in one spot; then move to a new spot nearby for an “undamaged” image

Adjusting FFT (as needed) - click on xxx

Changing grids

  1. Click camera in to move the camera back out of the beam

  2. Lower Mag to ~1000x

  3. Reduce beam intensity to ~50%

  4. Under Search tab of microscope control panel, click XY to reset stage

  5. Close column valves the most important thing!

Leaving the microscope

  1. Do Changing grids protocol

  2. Remove the holder from the microscope

  3. Remove your sample from the holder

  4. Re-insert the empty sample holder

  5. If no one is signed up to use the microscope within an hour, turn Filament off.

  6. Leave High Tension on and column valves closed

  7. Log your time on the e-logger and the paper log

  8. Transfer your images from the Windows server to Google Drive

Turning off the microscope if after 5 o’clock

  1. Turn off filament; wait about 2 min

  2. Remove center aperture (move objective lever on microscope to right)

  3. Turn off high tension

  4. Lift up styro mattress to protect equipment from liquid nitrogen

  5. Swap small styro dewer in for taller metal liquid N2 dewer (to catch braid)

  6. Press cryocycle button

  7. Turn off all 3 monitors

  8. Close locked doors behind you.